Immunomodulatory properties of conjugated linoleic acid

¹ From Loders Croklaan, Lipid Nutrition, Channahon, IL (MO); the Nutritional Immunology and Molecular Nutrition Laboratory, Department of Human Nutrition, Foods, and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA (JB-R); and Loders Croklaan, Lipid Nutrition, Wormerveer, Netherlands (ICMM)

² Presented at the workshop "The Role of Conjugated Linoleic Acid in Human Health," held in Winnipeg, Canada, March 13-15, 2003.

³ Address reprint requests and correspondence to M O’Shea, Loders Croklaan, Lipid Nutrition, 24708 West Durkee Road, Channahon, IL 60410. E-mail: marianne.o'shea{at}croklaan.com.

ABSTRACT

In vitro studies of the use of immune cells and animal models demonstrate that conjugated linoleic acid (CLA), a lipid, modulates immune function. In addition, recent publications demonstrate that 2 active CLA isomers (ie, cis-9,trans-11 CLA and trans-10,cis-12 CLA) modulate immune function in humans. Aspects of both the innate and adaptive immune responses are affected by dietary CLA supplementation. CLA consists of a mixture of isomers, which reduced immune-induced wasting and enhanced ex vivo lymphocyte proliferation in broilers and decreased tumor necrosis factor (TNF-) and interleukin 6 (IL-6) production in rat models. In mice, ex vivo lymphocyte proliferation and IL-2 production were increased. Furthermore, evidence suggests that the cis-9,trans-11 and trans-10,cis-12 CLA isomers exert distinct effects on immune function. Specifically, these 2 isomers have differential effects on specific T cell populations and immunoglobulin subclasses in animal and human studies. Herein, a systematic review of the literature and relevant new data are presented with an aim to compare data and to present an overview covering the innate and adaptive components of the immune response that are regulated by CLA. In addition, potential mechanisms of action are discussed and the need for future studies on the immunomodulatory properties of CLA are outlined in detail. The understanding of the mechanism(s) by which CLA increases immune function will aid in the development of nutritionally based therapeutic applications to augment host resistance against infectious diseases and to treat immune imbalances, which result in inflammatory disorders, allergic reactions, or both.

Key Words: Innate immunity • adaptive immunity • lymphocytes • macrophages • immunoglobulins

INTRODUCTION

Conjugated linoleic acid (CLA) refers to a class of positional and geometric conjugated dienoic isomers of linoleic acid (LA), of which cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) CLA predominate. CLA is naturally present in milk and meat of ruminants and can be produced industrially by partial hydrogenation of LA (1, 2). The concentrations of CLA in ruminant-derived products range from 3 to 7 mg CLA/g fat, depending on the source and processing of foods (1, 3-5). Estimated average daily intake of CLA from these dietary sources ranges from 0.19 to 1.5 g CLA (6, 7) and varies for different countries (2).

Most of the physiologic effects of CLA were observed after feeding animals with mixtures of CLA isomers that contain mostly c9,t11-CLA and t10,c12-CLA in roughly equal amounts. The most abundant CLA isomer in nature is c9,t11, amounting to as much as 90% of the total CLA content of dairy foods (8). Several reports show an association between these 2 isomers and protection against cancer (9-15), atherosclerosis (16, 17), alteration of body composition (18, 19), and diabetes (20). In addition, the results of studies that use animal models suggest that CLA enhances immune function (21-27) while ameliorating immune-mediated catabolism (25-30).

Furthermore, CLA stimulated production of anti-inflammatory cytokines and altered the concentration of insulinlike growth factor in bone tissues, which led to increased bone formation (31) and muscle mass (29, 32) and decreased subcutaneous fat (33).

Evidence suggests that individual CLA isomers could have different health effects (34). Differences in effects on immunoglobulin subclasses were observed; lymphocytes isolated from mice fed t10,c12-CLA produced more immunoglobulin A (IgA) and IgM but not IgG compared with controls or c9,t11-CLA-fed mice (35). The differential effect of 2 isomeric mixtures of CLA a 50:50 and 80:20 of c9,t11:t10,c12 CLA, respectively, was observed in humans, whereby the 50:50 mixture increased antigen-specific antibody concentration compared with the 80:20 mixture (36). Yamasaki et al (35) also found that the cis-9,trans-11 CLA isomer was involved in the enhancement of CD8⁺ T cells. Further differential effect of 2 isomeric mixtures of CLA at 50:50 and 80:20 of c9,t11:t10,c12 CLA, respectively, was observed in humans, whereby the 80:20 mixture significantly enhanced peripheral blood lymphocyte proliferation in response to the T cell mitogen phytohemagglutinin, whereas treatment with the 50:50 mixture significantly decreased concanavalin A (Con A)-induced blastogenesis (37).

The first human trial that assessed effects of a CLA mixture with the 2 active isomers present in relatively low amounts on immune function found no effect on the response to influenza vaccination or on any of the other immune functions measured (38, 39). This result is in contrast to another study, which provided evidence that a CLA mixture (mainly containing the 2 active isomers) has beneficial effects on immune function in humans as reflected by an increased seroprotection rate after vaccination with hepatitis B (37). Furthermore, new data now indicate further beneficial effects of CLA on immune responses in humans (HJ Song, ICM Mohede, D Rotondo, I Grant, KWJ Wahle, unpublished observation, 2003).

This review links the data from these human studies that investigate the immunomodulatory effects of CLA and studies that examine the mechanism(s) by which CLA exerts this effect. Future work in this area could lead to immune-enhancing products such as supplements and new CLA-enriched functional foods aimed at ameliorating the negative effects of immune imbalances such as allergies, inflammatory responses [eg, inflammatory bowel disease (IBD)], and prevention of infection and increased resistance for both immunocompromised and healthy individuals.

THE IMMUNE RESPONSE

The human immune response consists of 2 closely connected defense mechanisms, the innate and the adaptive immune system. The innate immune system consists of barriers, such as skin and mucosal surfaces (eg, intestinal, respiratory, and genitourinary tracts) and of broad pattern recognition that leads to phagocytosis and extracellular killing of invading agents. The adaptive immune system consists of B and T lymphocytes, which elicit their effector functions in an antigen-specific fashion. Development of effector cells is driven by the action of T helper (Th; CD4⁺) cells, which can be divided on the basis of the cytokines they produce into Th1 [interferon- (IFN-), interleukin 2 (IL-2)] and Th2 (IL-4, IL-5, IL-10) cells. After antigenic stimulation, a subset of lymphocytes become memory cells, which can elicit faster and more potent immune responses at a subsequent exposure with the same antigen (40). Lymphocytes and natural killer cells are derived from undifferentiated, self-renewing hemopoietic stem cells through highly regulated differentiation and maturation processes. These processes are mediated by microenvironmental factors, including cell-to-cell interactions, cytokines, and growth factors. With regard to the effects of CLA on immune cell development, phenotypic analysis of T cell subsets in the thymus and peripheral blood revealed that CLA acted first on immature thymocytes (ie, day 35 of dietary supplementation) and later modulated mature peripheral blood T cells (ie, days 49 to 72 of dietary supplementation) in a pig model (26). These findings suggest that CLA could influence critical pathways of thymocyte differentiation.

The purpose of this article is to discuss the effects of CLA on 1) mediators of immunity in the presence or absence of immune stimulation and its consequences on immune cell responsiveness on subsequent antigenic challenges, 2) regulation of innate and adaptive immune responses, and 3) amelioration of the negative effects because of abnormal disturbed immune reaction, inflammatory response, or both.

MEDIATORS OF IMMUNITY AFFECTED BY CONJUGATED LINOLEIC ACID

In vitro and ex vivo studies
CLA modulated eicosanoid production in both in vitro and ex vivo studies. Eicosanoid production is determined largely by the availability of substrate fatty acids in the membrane phospholipids and is also affected by the relative amounts of n–3 and n–6 polyunsaturated fatty acids (PUFAs) (41). In this regard, Sugano et al (42) showed that CLA down-modulated the release of leukotriene B₄ (LTB₄) from exudate cells (0.5 and 1 g/100 g for 3 wk). CLA did not affect the release of histamine. Additionally, a dose-response relation was observed in splenic LTB₄, lung LTC₄, and serum prostaglandin E₂ (PGE₂) concentrations (all had significant differences between control and 1 g/100 g CLA groups). The researchers stated that the reduction by CLA of the concentrations of n–6 PUFAs in peritoneal exudate cells and splenic lymphocyte total lipids was responsible, in part, for the reduced eicosanoid concentrations. Li and Watkins (31) evaluated the effects of CLA on the composition of tissue fatty acids and ex vivo PGE₂ production in rats fed diets varying in n–6 and n–3 PUFAs. Four groups of rats were given a basal semipurified diet containing added fat (7 g/100 g) for 42 d. The fat treatments were formulated to contain CLA (0 compared with 1 g/100 g diet) and different ratios of n–6 soybean oil (SBO with an n–6:n–3 of 7:3) and n–3 fatty acids menhaden oil and safflower oil mixture (MSO with an n–6:n–3 of 1:8). Fatty acids in liver, serum, muscle, heart, brain, spleen, and bone were analyzed by capillary gas-liquid chromatography. CLA isomers were found in all rat tissues that were analyzed, although their concentrations varied. Dietary CLA decreased inter alia the concentrations of n–6 PUFAs but increased the concentrations of n–3 PUFAs in the tissues that were analyzed. Ex vivo PGE₂ production in bone organ culture was decreased by n–3 PUFAs and CLA.

The CLA mixture and the individual isomers were found to inhibit resting production of ¹⁴C-PGF₂a by 50%, 43%, and 40%, respectively, in human saphenous vein endothelial cells (43). A dose-dependent inhibition of stimulated ¹⁴C-prostaglandins was observed with the CLA mixture (concentration that inhibits 50%, 100 µmol/L). The c9,t11- and t10,c12-isomers (50 µmol/L) individually inhibited the overall production of stimulated ¹⁴C-prostaglandins (between 35% and 55% and 23% and 42%, respectively). The overall degree of ¹⁴C-arachidonic acid (AA) incorporation into membrane phospholipids of the CLA (mixture and individual isomers) treated cells was found to be lower than that of control cells, and the c9,t11-isomer was found to increase the incorporation of ¹⁴C-AA into phosphatidylcholine. Docosahexaenoic acid, eicosapentaenoic acid, and LA did not alter the overall degree of incorporation of ¹⁴C-AA (43).

With use of an ex vivo design, Turek et al (21) also investigated whether CLA altered cytokine and eicosanoid production in a similar manner as n–3 PUFAs. The extent of CLA incorporation into a lymphoid organ (spleen) was determined by fatty acid analysis, and peritoneal macrophage tumor necrosis factor- (TNF-), IL-1, and IL-6 amounts and liver homogenate PGE₂ biosynthesis were measured in rats fed varying ratios of n–6 and n–3 PUFAs with CLA (c9,t11- and t10,c12-isomers) and without CLA. During a 42-d period, 4 groups of 6 male weanling Sprague-Dawley rats were fed one of the following diets: SBO (diet rich in n–6 fatty acids), SBO and CLA (1 g/100 g), menhaden oil and safflower oil mixture (MSO; rich in n–3 fatty acids), or MSO and CLA (1 g/100 g). The MSO diet significantly reduced liver PGE₂ production compared with the amounts from SBO-fed rats. This reduction was expected on the basis of the results of other research, which showed that n–3 PUFAs reduce PGE₂. CLA caused a slight reduction in mean liver PGE₂ production. Although this reduction was not significant, the effect was greater for SBO-fed rats. These findings suggest that as the dietary ratio of n–6 to n–3 PUFAs increases, the magnitude of the modulatory effect of CLA on PGE₂ production is also greater. CLA significantly reduced basal macrophage TNF- production in the control medium from rats fed either SBO (42% decrease) or MSO (54% decrease). CLA significantly reduced mean basal IL-6 amounts in macrophage control medium from SBO-fed rats (79.6% decrease). On the basis of these data, the researchers stated that the dietary profiles of PUFAs do not affect CLA reduction of basal TNF amounts but do influence the effect of CLA on basal IL-6 production and liver PGE₂ production. The data also indicated that there is some interaction between the type of PUFAs and CLA in eicosanoid regulation of cytokine production (21).

To evaluate whether CLA feeding affects immunoglobulin production, Sprague-Dawley rats were fed a diet supplemented with CLA at concentrations of 0.5 g/100 g and 1 g/100 g for 3 wk (41, 42). The spleen and mesenteric lymph node were excised and lymphocytes were prepared. After 24 h of incubation, the concentrations of different immunoglobulins in the culture supernatant were measured by enzyme-linked immunosorbent assay. Results showed that the concentrations of IgA and IgM in mesenteric lymphocytes from rats fed 1 g/100 g CLA were 3 and 50 times higher, respectively, than concentrations in lymphocytes from control rats. IgG was detected in the CLA-fed group but not in the control rats. The concentration of IgG in the 1 g/100 g CLA group was 9 times higher than in the 0.5 g/100 g CLA group. CLA feeding significantly raised serum concentrations of IgA, IgM, and IgG but decreased concentrations of IgE, particularly in rats fed 1 g/100 g CLA (42). Hence, CLA differentially regulates class-specific production of immunoglobulin.

Spleen lymphocytes isolated from the mice fed diets containing 1% t10,c12-CLA for 3 wk produced more IgA from unstimulated spleen lymphocytes in the t10,c12-CLA group than in controls (35). Conversely, 1% c9,t11-CLA did not affect the production of any of the immunoglobulin subclasses. The proportion of B cells in the spleen lymphocyte population was significantly lower in the c9,t11-CLA group and higher in the t10,c12-CLA group than in the controls. These results suggest that c9,t11- and t10,c12-CLA can stimulate different immunologic effects (35).

: CLA modulates the immune system and prevents immune-induced wasting (29). Cook et al (44) used 1-d-old chicks and fed them LA (95% pure) or CLA (95%) containing 43% c9,t11 and 44% t10,c12 as the predominant CLA isomers. Half of the chicks in each dietary treatment group were injected with sterile buffered saline; the other half received 1 mg/kg body weight endotoxin consisting of lipopolysaccharide (LPS) from Escherichia coli. The change in body weight over the following 24 h was monitored. Chicks fed CLA gained weight after endotoxin injection, whereas chicks fed the control diet either lost weight or failed to grow. Additional experiments were conducted in rats to determine whether the protection against immune-induced wasting was conserved across animal species. Rats were also responsive to the protective effects of CLA against immune stress. Sugano et al (42) concluded that CLA not only enhanced specific immune responses but also mitigated the adverse effects of the immune system such as immune-induced wasting. The previous findings were further substantiated in mice fed 0.5 g/100 g CLA in the diet; the CLA group was protected against body weight wasting and feed intake after LPS-injection compared with controls fed corn oil ( Human studies
In a recent randomized, double-blind, placebo-controlled study the effects of dietary CLA supplements on immune function of healthy men and women (n = 28) aged 1845 without immune challenge were investigated (Song et al, unpublished observation, 2003). Subjects were randomly divided into 2 groups receiving placebo high-oleic sunflower oil or CLA (50:50 mixture of c9,t11:t10,c12 isomers). Subjects took 6 capsules (3 g high-oleic sunflower oil or CLA) per day for 12 wk. Blood samples were taken at baseline, at 6 and 12 wk during supplementation, and after a 12-wk washout period and analyzed for cytokines (IL-10, IFN-, TNF-), immunoglobulins (IgA, IgG, IgM, and the allergy-related immunoglobulin, IgE). In peripheral blood mononuclear cells, CLA decreased TNF- and IFN- at 12 wk by 20% (P < 0.05) and 40% (P < 0.05), respectively, and at washout by 40% (P < 0.05). CLA increased IL-10 by 20% and 40% for 6 (P < 0.05) and 12 (P < 0.01) wk, respectively. Placebo also increased IL-10 at 12 wk and at washout (P < 0.05). Plasma IgA was increased 10% at 12 wk (P < 0.05) but decreased by placebo at week 6 (20%; P < 0.01). IgM was increased at 6 wk (P < 0.01), 12 wk (P < 0.01), and at washout (P < 0.01); placebo also increased IgM at 12 wk (P < 0.05). IgE was decreased at 12 wk (10%) and by 20% at washout (P < 0.01); placebo increased values at 6 wk by 20% (P < 0.05). The suppressed IFN- and enhanced IL-10 production suggest that CLA could be beneficial in certain types of allergic or inflammatory responses (eg, IBD (Song et al, unpublished observation, 2003).

Conclusions on conjugated linoleic acid and immunomodulation
Compelling evidence is available, which demonstrates the ability of CLA to modify soluble factors or mediators of immunity such as eicosanoids, prostaglandins, cytokines, and immunoglobulin production. The data from Song et al (unpublished data, 2003), demonstrating an increase in concentrations of specific IgA and IgM, coupled with a decrease in the allergy-related immunoglobulin IgE in humans, is consistent with the findings from Sugano et al (42) in rats. The reduction in IgE suggests an antiallergic potential of CLA. IgE has a central role in type-I (or IgE-mediated) allergic reactions, eg, to airborne and food allergens (ie, pollen, soybean proteins, wheat proteins, peanuts). IgE regulates the release of chemical mediators such as histamine and leukotrienes by mast cells and basophils, resulting in allergic disorders.

IgE is formed during a Th2-mediated immune response. Antigen-specific, Th cells stimulate the B lymphocytes to produce and release antibodies (or immunoglobulins) that bind to specific antigens. Th1 cells stimulate the production of antibodies of IgG2a isotype, whereas Th2 cells induce the production of IgG1 and IgE antibody isotypes by B cells. We propose that CLA regulates the balance between Th1 and Th2 responses. This hypothesis is supported by the fact that a reduction in IgE concentrations was observed after feeding with CLA. This decrease in IgE was accompanied by an increase in IgM, IgA, and IgG. IgM is frequently associated with the immune response to antigenically complex, bloodborne infectious agents and is, therefore, used as a diagnostic screening antibody. IgA can serve as antiallergic factors interfering with intestinal absorption of allergens; furthermore, plasma IgA amounts correlate well with those seen in the intestine. IgG also functions as an antiallergic factor by competing with the allergen-specific IgE to bind to the receptor on the surface of the target cells such as mast cells and basophils. Therefore, both IgA and IgG exert a preventive effect on IgE-mediated allergy and viral infections (37). Evidence suggests that the t10,c12-CLA isomer is responsible for the effects on immunoglobulins and not the c9,t11-CLA isomer (35).

The decreased production of proinflammatory cytokines such as TNF- in both human and animal models by CLA (Song et al, unpublished observation, 2003; 21) suggests that the immune status is channeled into an anti-inflammatory profile. TNF- has a central role in the inflammatory response and is a key mediator in many chronic immunopathologies, including cachexia, atherosclerosis, cancer, obesity, and rheumatism. In addition to the down-regulation and improvement of adverse effects that can occur during immune stimulation as seen with the prevention of endotoxin-induced cachexia in chickens (44), CLA has implications for the treatment of inflammatory conditions such as IBD. In this regard, the decreased IFN- production observed in CLA-fed humans was first reported in the colonic mucosa of pigs with bacterially induced colitis. With use of a pig model of IBD, Hontecillas et al (30) demonstrated that CLA ameliorated tissue inflammation and weight loss associated with IBD. Similar clinical and pathologic findings were observed in mouse models of IBD (R Hontecillas and J Bassaganya-Riera, unpublished observation, 2003; 45). Future investigations into the therapeutic potential of CLA against inflammatory conditions such as allergies and IBD is warranted.

REGULATION OF INNATE RESPONSES BY CONJUGATED LINOLEIC ACID

The cells of the innate immune response such as monocytes or macrophages and natural killer cells secrete cytokines (TNF-, IL-1, IL-6), prostaglandins, and leukotrienes early during the nonspecific immune response. They give direction and contribute to the specific immune response and associated inflammatory reaction. Prostaglandins have local effects, acting mainly in the tissues where they are synthesized. In addition, they can activate immune cells to produce specific cytokines. Leukotrienes mediate many of the inflammatory phenomena that are characteristic of immediate hypersensitivity reactions.

In vitro and ex vivo studies
When murine keratinocytes (HEL-30) were preincubated with AA and either LA or CLA and challenged with 12-O-tetradecanoyl-phorbol-13-acetate (tumor promotor), the amount of PGE₂ in cells that were pretreated with CLA was significantly lower than in cells pretreated with LA (41).

Turek et al (21) demonstrated that the ex vivo addition of CLA into rat macrophage cultures significantly reduced basal TNF- production (42% decrease). In vitro studies in a macrophage cell line demonstrated that the c9,t11-CLA isomer was responsible for the inhibition of LPS-induced TNF- production (46). Furthermore, peritoneal macrophages from CLA-fed mice (0.5% of the diet) produced less nitric oxide when compared with cells from control-fed animals on in vitro exposure to IFN- and LPS. Isolated splenocytes from CLA-fed animals had decreased IL-4; however, when stimulated with Con A for 44 h, IL-2 and the IL-2 to IL-4 ratio were elevated (46).

In vivo studies
In addition to dietary supplementation evidence now suggests that topical application of CLA could have antiallergic properties against anaphylaxis and allergic pruritus in mice (47). Inhibitory effects of dietary CLA were observed on the immediate type 1 hypersensitivity reaction, with CLA significantly suppressing the decrease in blood pressure and blood flow induced by the hen egg-white lysozyme in a mouse model of anaphylaxis. After oral administration, CLA showed antipruritic activity, with significant inhibition of scratching behavior induced by compound 48/80, a histamine-release agent. When painted onto the skin, CLA also inhibited compound 48/80, platelet-activating factor, protease-induced scratching behavior, and compound 48/80-induced vasodilation of the skin (47).

Further work has now demonstrated that CLA significantly reduced the harmful effects of intranasally administered influenza virus in rats (Loders Croklaan, personal communication, 2003). CLA was administered at 1% (by wt) of the diet for 4 wk before infection with rat-adapted influenza virus and for 2 wk thereafter, at which point the animals were killed. A subset of animals from each group (n = 10) was killed on day 2 after influenza infection to monitor effects of CLA on the early or innate responses. CLA-fed animals had significantly lower peritoneal fat mass and higher lean body mass at day 2 after influenza infection compared with the control group. CLA-fed animals showed significantly enhanced relative lung and spleen weights (P < 0.05) compared with the control group on day 2, indicative of the initial innate response to infection. In addition virus particles as measured by mRNA were significantly reduced in the CLA-fed group 2 d after infection compared with the control group. Mortality rate was reduced by 22% in the CLA-fed group. These findings suggest that CLA has a protective effect against influenza infection.

Conclusions on conjugated linoleic acid and innate responses
CLA in the diet appears to affect different immune-related mechanisms involved in both allergic reactions and infections. That CLA reduces PGE₂ and IL-12 secretion as monocyte or macrophage product is theoretically an important step in reducing the chance of a Th2-mediated response and thereby the chance of a new allergic reaction. Also observed in this study was the decrease in leukotriene and prostaglandin production in organs from CLA-fed animals. Leukotrienes mediate many of the inflammatory phenomena that are characteristic of immediate hypersensitivity reactions such as direct effects on smooth muscle cells and blood vessels contributing to the recruitment of inflammatory cells.

The effects of CLA on aspects of the innate response to viral infection such as increased organ weight at the sight of infection because of increased production and recruitment of inflammatory cells coupled with the reduction in viral particles in CLA-fed animals indicate a reduction in the adverse effects resulting from influenza infection. The increased lean body mass observed in this model because of pretreatment with CLA for 5 wk before infection could enhance the ability of the animal to respond more effectively to the influenza challenge. However, at this point human data are lacking; therefore, it is premature to speculate the effects of CLA as a treatment for relieving the effects of influenza and other viral infections in humans. These animal data are promising and a human study is warranted.

AMELIORATION OF THE NEGATIVE EFFECTS OF ABNORMAL IMMUNE AND INFLAMMATORY RESPONSE

In vitro and ex vivo studies
Isolated spleen lymphocytes from mice fed c9,t11-CLA, t10,c12-CLA, or a mixture of both isomers at 1% of the diet demonstrated differential effects of the individual isomers on Con A-stimulated immunoglobulin production (35). Spleen lymphocytes isolated from mice fed t10,c12-CLA produced more IgA and IgM but not IgG compared with controls. Conversely, c9,t11-CLA did not affect production of any of the immunoglobulin subclasses. Lymphocytes isolated from c9,t11-CLA-fed animals produced more TNF- than the control group. The proportion of B cells in the spleen lymphocyte population was significantly lower in the c9,t11-CLA-fed group and higher in the t10,c12-CLA group compared with controls. The T cell populations also differed between groups with the percentage of CD4⁺ T cells lower in the t10,c12-CLA group and the percentage of CD8⁺ T cells higher in the c9,t11-CLA-fed group compared with controls. The CD8⁺ T cells were higher in the group receiving the mixture, and the ratio of CD4⁺ to CD8⁺ was reduced. These data clearly demonstrate differential effects of both isomers on the immune system in mice and simultaneous intake of the 2 isomers can change the T cell population (35). In addition, the increase in CD8⁺ T cells is consistent with the results of previous studies in pig models of immunomodulation (25-27).

With regard to the modulation of the humoral immune response by CLA, a similar effect was observed on lymphocytes isolated from Sprague-Dawley rats fed a diet supplemented with CLA at concentrations of 0.5 and 1 g/100 g for 3 wk (42, 44). CLA feeding enhanced production of IgG in spleen lymphocytes stimulated by LPS. The amount of IgG was 1.3 times higher in the 1 g/100 g CLA group than in the control group.

The modulating effect of CLA on immune mediators was also shown by Whigham et al (48) in an ex vivo experiment. Those researchers investigated the role of CLA in type-I (immediate) hypersensitivity with use of a guinea pig tracheal superfusion model for measuring antigen-induced contraction of airway smooth muscle and inflammatory mediator release. Female Hartley guinea pigs were fed a diet supplemented with 0.25 g corn oil or 0.25 g LA/100 g diet (control) or 0.25 g CLA/100 g diet for at least 1 wk before and during active sensitization to ovalbumin antigen. Trachea from sensitized guinea pigs were suspended in air-filled water-jacketed (37 °C) tissue chambers in a superfusion apparatus. Tracheae were superfused with buffer that contained antigen, and tissue contraction was recorded. Superfusate was collected at 90-sec intervals for evaluation of histamine and PGE₂ release. CLA significantly reduced antigen-induced histamine and PGE₂ release of some inflammatory mediators during type-I hypersensitivity reactions.

Further work from this group demonstrated that CLA reduced ex vivo antigen-induced eicosanoid release from lung, trachea, and bladder of guinea pigs fed 0.25% CLA (49). Thromboxane B₂, 6-keto-PGF₁, PGF₂, and PGE₂ were reduced by 57–75% in lung, 45–65% in trachea, and 38–60% in bladder, and LTC₄, LTD₄, and LTE_e were reduced by 87%, 90%, and 50% in lung, trachea, and bladder, respectively_. CLA did not affect basal mediator release after antigen challenge; these inflammatory mediators are products of cyclooxygenase and lipoxygenase enzymes. Although CLA was shown not to affect cyclooxygenase and lipoxygenase protein amounts, it is postulated that this effect could be mediated by enzyme inhibition or alteration of phospholipid composition, thereby reducing the precursors required for generation of eicosanoid products by cyclooxygenase and lipoxygenase during an inflammatory response (49).

In vivo studies
Sugano et al (41) demonstrated that CLA stimulated the proliferation of spleen lymphocytes previously stimulated by pokeweed mitogen. CLA also enhanced the production of IL-2 after 3 or 6 wk of feeding Balb/c mice with 0.1% of weight, 0.3 g CLA/100 g diet, or 0.9 g CLA/100 g diet, thus suggesting an improvement of the proliferative abilities of lymphocytes (41). Chew et al (50) demonstrated the effect of CLA on the cellular immune system in an in vitro design; in combination, CLA and ß-carotene boosted in vitro proliferation of lymphocytes (in blood, peritoneum) that were stimulated by pokeweed mitogen, Con A, or phytohemagglutinin. However, CLA inhibited the production of IL-2 by lymphocytes and also phagocytotic activity of macrophages. When CLA and ß-carotene were added together, spontaneous proliferation and cytotoxic activity of lymphocytes were both enhanced (41).

The enhancement in cytotoxic activity observed by Chew et al (50) in vitro was demonstrated in vivo with use of a pig model. Specifically, CLA fed to pigs at 1.33 g/100 g diet for 42 d modulated phenotype and effector functions of porcine CD8⁺ lymphocytes, inducing in vivo expansion of T cell receptor-ßCD8, natural killer cells, and T cell receptor-ßCD8ß cells, as well as cytotoxicity of specific CD8⁺ mediator functions as measured by granzyme activity (26). CLA enhanced cellular immunity by modulating phenotype and effector functions of CD8⁺ cells involved in both adaptive and innate immunity (26).

With use of a similar porcine model with longer-term feeding of CLA (1.33 g/100 g diet) for 75 d, Bassaganya-Riera et al (27) demonstrated that CLA enhanced the ability of CD8⁺ T cells to proliferate when stimulated with viral antigens. The in vivo expansion of CD8⁺ T cells persisted after CLA was withdrawn from the diet (67 d), whereas effector functions (antigen-stimulated proliferation and cytotoxicity) disappeared earlier (25 d). These findings demonstrate that the effects of CLA persist beyond the period of dietary supplementation (27).

A recent study examined the effects of CLA on viral infectivity in a pig model of virally induced immunosuppression (51). After infection with type-2 porcine circovirus (PCV2), we examined the effect of CLA on the development of lesions (ie, lymphoid depletion and pneumonia) and the kinetics of cellular and antibody responses against PCV2. The infection with PCV2 induced a depletion of B cells, which was more accentuated in pigs fed the control diet, in which IL-2 mRNA expression was down-regulated. The histopathologic examination of the lungs revealed that the interstitial pneumonia tended to be more severe in infected pigs fed the control diet, which were also affected by growth retardation (51). These histopathologic improvements correlated with greater numbers of CD8⁺ T cells in PCV2-infected pigs fed CLA (51).

Human studies
Although promising effects of CLA are observed in animals in vitro, ex vivo, and in vivo studies, clear evidence for a beneficial health effect of CLA in humans is limited. A human in vivo study (36) showing a CLA-induced effect on the humoral immune system was published. In the study by Albers et al (36), a promising infection model was used to measure the effects of CLA on the immune system. In that model, the main dynamic immunologic parameters of the humoral and cellular immune response after hepatitis B (HB) vaccination were investigated. The humoral immune response is reflected in the measured seroprotection rate [the number of subjects with serum anti-HB antibody concentrations >10 IU/L (known to be the protective concentration) compared with the number of subjects with serum anti-HB antibody concentrations <10 IU/L] and HB antibody concentrations. The cellular immune response is reflected in the delayed-type hypersensitivity test. In a 12-wk, randomized, placebo-controlled, double-blind trial, Albers et al (36) investigated the immunomodulating effect of CLA in 71 subjects (healthy men, aged 30–70 y). They were randomly assigned to CLA consisting of 50% c9,t11 and 50% t10,c12, CLA consisting of 80% c9,t11 and 20% t10,c12, or sunflower oil fatty acids (placebo). A dosage amount of 3 g/d in soft-gel capsules provided 1.7 g CLA/d in the treatment groups. At baseline and after 12 wk in vivo, cell-mediated immune responsiveness was measured. The results of that study indicated an increased response after HB vaccination that was reflected in HB-specific antibody titers and seroprotection rate. That study is the first in which CLA was shown to stimulate the humoral immune response in humans as reflected in an increased seroprotection rate after vaccination.

The previous results are in contrast to the study by Kelley et al (38) in which 10 women were supplemented with 3.9 g CLA/d over 63 d, and no alterations of indexes of immune status after an influenza vaccination or a delayed-type hypersensitivity test were reported. This apparent discrepancy could be explained by a different isomer composition of the CLA test product. Specifically, the amount of active isomers, c9,t11 and t10,c12, was very low and the trans,trans isomer of CLA was high in the product that the researchers used. In addition, the statistical power of that study was limited because of the reduced number of subjects in the experiment.

Regulation of adaptive immunity by conjugated linoleic acid: conclusions
The data from these studies demonstrate an improvement in antigen-specific effector functions of both cellular and humoral responses to bacterial and viral antigens associated with dietary CLA supplementation both in animal models and humans. PGE₂ concentrations were reduced by CLA in a type-I allergy model in guinea pigs (48). PGE₂ plays a crucial role in type-I (or IgE-mediated) allergic reactions resulting from the development of Th2 cells. Further studies are warranted to investigate the promising effects of CLA on hypersensitivity reactions.

Because CLA increased the number of human subjects mounting a protective response without further stimulating ongoing responses, CLA supplementation could be particularly beneficial for immunocompromised individuals who are slow or low responders to the vaccination or challenge (36). This hypothesis suggests that CLA could have properties as an oral adjuvant to vaccination and could have particular relevance for the elderly population incapable of mounting a sufficient response to vaccination to confer immune protection. It remains to be established whether similar benefits can be achieved after exposure to naturally occurring infections as seen in animal models of infectious disease as discussed previously. Dietary CLA supplementation could be used as an immunotherapeutic agent designed to counter some of the deleterious effects of declined immune function as a result of stress, exercise, or aging. The hypothesis that CLA can ameliorate the deleterious effects of immune senescence warrants testing in larger-scale longer-term intervention trials designed to assess the incidence and severity of naturally occurring infections in control and CLA-fed subjects.

GENERAL CONCLUSIONS AND POSSIBLE MECHANISMS OF ACTION

Several mechanisms are proposed as underlying the modulating effects of CLA on the immune system. With focus on the effects of basal mediators of immunity discussed earlier, it was hypothesized by Pariza et al (52) that CLA could have a modulating effect on TNF- by altering eicosanoid signaling. Altered eicosanoid signaling could, in turn, affect a range of biological activities, such as cytokine synthesis and immune functions, including antigen presentation. This hypothesis (52) is in agreement with that of Li and Watkins (31) with regard to the potential mechanism by which dietary CLA exerts its chemoprotection. In that study, it was suggested that CLA inhibits PGE₂ synthesis with likely consequences on the modulation of immune cells. CLA supplementation at 1.7 g/d in humans led to a significant (P < 0.05) increase in c9,t11-CLA incorporation into cell membranes of isolated peripheral blood mononuclear cells compared with control subjects (36). Modification of the cell membrane has implications for subsequent eicosanoid production and cell signaling events. Cell-to-cell contact is critical during the development of T and B cell effector functions. This mechanism, therefore, could also be responsible for the many immunomodulating effects of CLA.

An alternative or complementary hypothesis is that CLA interacts with peroxisome proliferator-activated receptors (PPARs). PPARs (, /ß, and ) are fatty acid receptors that regulate the expression of genes involved in energy homeostasis and immune function (53). PPARs bind to the PPAR-response element and repress or induce the transcription of target genes. The resultant change in gene expression accounts for effects on lipid metabolism, as well as on energy balance, thermogenesis, glucose metabolism, and the atherosclerotic and carcinogenic processes (54). The CLA isomers are potent modulators of PPARs (20, 55). These fatty acid receptors are widely expressed in the immune system and are involved in regulating expression of various genes involved in proliferation of lymphocytes and monocytes or macrophages, apoptosis, and inflammation. In similar action to CLA, synthetic PPAR- agonists are known to inhibit the production of proinflammatory cytokines, including TNF- in monocytes (56), in addition to affecting the differentiation of monocytes and macrophages (57). Antigen presentation is one of the principal functions of these cells; therefore, modification of this function has implications in the development of subsequent effector functions in a Th1 or Th2 response. Therefore, modulation of cells at the antigen presentation amount by CLA can affect subsequent cellular and humoral responses to antigen challenge, thus affecting aspects of both adaptive and innate responses.

Definitive molecular evidence in vivo on the mechanism(s) of action of CLA is not available. The use of tissue-specific PPAR--deficient mice could provide the most definitive molecular evidence in vivo of the mechanism of action of CLA. Further investigations of the downstream effects of CLA on the differential modulation of PPAR expression/function and eicosanoid pathways will provide key insights to possible preventive and therapeutic applications.

ACKNOWLEDGMENTS

The authors had no conflicts of interest.

REFERENCES

1. Chin SF, Storkson JM, Liu W, Albright KJ, Pariza MW. Conjugated linoleic acid (9,11- and 10,12-octadecadienoic acid) is produced in conventional but not germ-free rats fed linoleic acid. J Nutr1994;124:694–701.
2. Parodi PW. Conjugated linoleic acid: an anticarcinogenic fatty acid present in milk fat. Aust J Dairy Technol1994;49:93–7.
3. Shantha NC, Ram LN, O’Leary J, Hicks CL, Decker EA. Conjugated linoleic-acid concentrations in dairy products as affected by processing and storage. J Food Sci1995;60:695–7.
4. Lin H, Boylston TD, Chang MJ, Luedecke LO, Shultz TD. Survey of the conjugated linoleic acid contents of dairy products. J Dairy Sci1995;78:2358–65.
5. Shantha NC, Decker EA. Conjugated linoleic acid concentrations in processed cheese containing hydrogen donors, iron and dairy-based additives. Food Chem1993;47:257–61.
6. Piperova LS, Teter BB, Bruckental I, et al. Mammary lipogenic enzyme activity, trans fatty acids and conjugated linoleic acids are altered in lactating dairy cows fed a milk fat–depressing diet. J Nutr2000;130:2568–74.
7. Fritsche J, Steinhart H, Kardalinos V, Klose G. Contents of trans-fatty acids in human substernal adipose tissue and plasma lipids: relation to angiographically documented coronary heart disease. Eur J Med Res1998;3:401–6.
8. Chin SF, Liu W, Storkson JM, Ha YL, Pariza MW. Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognised class of anticarcinogens. J Food Compos Anal1992;5:185–97.
9. Belury MA, Nickel KP, Bird CE, Wu Y. Dietary conjugated linoleic acid modulation of phorbol ester skin tumour promotion. Nutr Cancer1996;26:149–57.
10. Ha YL, Storkson J, Pariza MW. Inhibition of Benzo(a)pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid. Cancer Res1990;50:1097–101.
11. Ha YL, Grimm NK, Pariza MW. Anticarcinogens from fried ground beef: heat/altered derivatives of linoleic acid. Carcinogenesis1987;8:1881–7.
12. Ip C, Banni S, Angioni E, et al. Conjugated linoleic acid-enriched butter fat alters mammary gland morphogenesis and reduces cancer risk in rats. J Nutr1999;129:2135–42.
13. Ip C, Chin SF, Scimeca JA, Pariza MW. Mammary cancer prevention by conjugated dienoic derivative of linoleic acid. Cancer Res1991;51:6118–24.
14. Ip MM, Masso-Welch PA, Shoemaker SF, Shea-Eaton WK, Ip CC. Conjugated linoleic acid inhibits proliferation and induces apoptosis of normal rat mammary epithelial cells in primary culture. Exp Cell Res1999;250:22–34.
15. Liew C, Schut HAJ, Chin SF, Pariza MW, Dashwood RH. Protection of conjugated linoleic acids against 2-amino-3-methylimidazo(4,5-f)quinoline-induced colon carcinogenesis in the F344 rat: a study of inhibitory mechanisms. Carcinogenesis1995;16:3037–43.
16. Lee KN, Kritchevsky D, Pariza MW. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis1994;108:19–25.
17. Nicolosi RJ, Rogers EJ, Kritchevsky D, Scimeca JA, Huth PJ. Dietary conjugated linoleic acid reduces plasma lipoproteins and early aortic atherosclerosis in hypercholesterolemic hamsters. Artery1997;22:266–77.
18. Park Y, Pariza MW. Evidence that commercial calf and horse sera can contain substantial amounts of trans-10, cis-12 conjugated linoleic acid. Lipids1998;33:817–9.
19. de Deckere EA, van Amelsvoort JM, McNeill GP, Jones P. Effects of conjugated linoleic acid (CLA) isomers on lipid levels and peroxisome proliferation in the hamster. Br J Nutr1999;82:309–17.
20. Houseknecht KL, Vanden Heuvel JP, Moya-Camarena SY, et al. Dietary conjugated linoleic acid normalizes impaired glucose tolerance in the Zucker diabetic fatty fa/fa rat. Biochem Biophys Res Commun1998;244:678–82.
21. Turek JJ, Li Y, Schoenlein IA, Allen KGD, Watkins BA. Modulation of macrophage cytokine production by conjugated linoleic acids is influenced by the dietary n–6:n–3 fatty acid ratio. J Nutr Biochem1998;9:258–66.
22. Hayek MG, Han SN, Wu D, et al. Dietary conjugated linoleic acid influences the immune response of young and old C57BL/6NCrlBR mice. J Nutr1999;129:32–8.
23. Wong MW, Chew BP, Wong TS, Hosick HL, Boylston TD, Shultz TD. Effects of dietary conjugated linoleic acid on lymphocyte function and growth of mammary tumors in mice. Anticancer Res1997;17:987–93.
24. Yang M, Pariza MW, Cook ME. Dietary conjugated linoleic acid protects against end stage disease of systemic lupus erythematosus in the NZB/W F1 mouse. Immunopharmacol Immunotoxicol2000;22:433–49.
25. Bassaganya-Riera J, Hontecillas R, Bregendahl K, Wannemuehler MJ, Zimmerman DR. Effects of dietary conjugated linoleic acid in nursery pigs of dirty and clean environments on growth, empty body composition, and immune competence. J Anim Sci2001;79:714–21.
26. Bassaganya-Riera J, Hontecillas R, Zimmerman DR, Wannemuehler MJ. Dietary conjugated linoleic acid modulates phenotype and effector functions of porcine cd8(+) lymphocytes. J Nutr2001;131:2370–7.
27. Bassaganya-Riera J, Hontecillas R, Zimmerman DR, Wannemuehler MJ. Long-term influence of lipid nutrition on the induction of CD8(+) responses to viral or bacterial antigens. Vaccine2002;20:1435–44.
28. Cook ME, Miller CC, Park Y, Pariza M. Immune modulation by altered nutrient metabolism: nutritional control of immune-induced growth depression. Poult Sci1993;72:1301–5.
29. Miller CC, Park Y, Pariza MW, Cook ME. Feeding conjugated linoleic acid to animals partially overcomes catabolic responses due to endotoxin injection. Biochem Biophys Res Commun1994;198:1107–12.
30. Hontecillas R, Wannemeulher MJ, Zimmerman DR, et al. Nutritional regulation of porcine bacterial-induced colitis by conjugated linoleic acid. J Nutr2002;132:2019–27.
31. Li Y, Watkins BA. Conjugated linoleic acids alter bone fatty acid composition and reduce ex vivo prostaglandin E-2 biosynthesis in rats fed n–6 or n–3 fatty acids. Lipids1998;33:417–25.
32. Park Y, Albright KJ, Liu W, Storkson JW, Cook ME, Pariza MW. Effect of conjugated linoleic acid on body composition in mice. Lipids1997;32:853–8.
33. Dugan MER, Aalhus JL, Schaefer AL, Kramer JKG. The effect of conjugated linoleic acid on fat to lean repartitioning and feed conversion in pigs. Can J Anim Sci1997;77:723–5.
34. Pariza MW, Park Y, Cook ME. The biologically active isomers of conjugated linoleic acid. Prog Lipid Res2001;40:283–98.
35. Yamasaki M, Chujo H, Hirao A, et al. Immunoglobulin and cytokine production from spleen lymphocytes is modulated in C57BL/6J mice by dietary cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid. J Nutr2003;133:784–8.
36. Albers R, van der Wielen RPJ, Brink EJ, Hendriks HFJ, Dorovska-Taran VN, Mohede ICM. Effects of cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid (CLA) isomers on immune function in healthy men. Eur J Clin Nutr2003;57:595–603.
37. Roche HM, Noone E, Nugent A, Gibney MJ. Conjugated linoleic acid: a novel therapeutic nutrient? Nutr Res Rev2001;14:173–87.
38. Kelley DS, Taylor PC, Rudolph IL, et al. Dietary conjugated linoleic acid did not alter immune status in young healthy women. Lipids2000;35:1065–71.
39. Kelley DS, Simon VA, Taylor PA, Rudolph IL, Benito P, Nelson GJ. Increased conjugated linoleic acid (CLA) concentration of peripheral blood mononuclear cells (PBMC), does not alter their function. FASEB J2001;15:A1094(abstr).
40. Griffiths M. Nonspecific protective mechanisms. Introduction to human physiology. 2nd ed. New York: Macmillan Publishing, 1981:331–53.
41. Sugano M, Yamasaki M, Yamada K, Huang YS. Effect of conjugated linoleic acid on polyunsaturated fatty acid metabolism and immune function. In: Yurawecz MP et al, ed. Advances in conjugated linoleic acid research. Vol 1. Champaign, IL: AOCS Press, 1999:327–39.
42. Sugano M, Tsujita A, Yamasaki M, Noguchi M, Yamada K. Conjugated linoleic acid modulates tissue levels of chemical mediators and immunoglobulins in rats. Lipids1998;33:521–7.
43. Urquhart P, Parkin SM, Rogers JS, Bosley JA, Nicolaou A. The effect of conjugated linoleic acid on arachidonic acid metabolism and eicosanoid production in human saphenous vein endothelial cells. Biochim Biophys Acta2002;1580:150–60.
44. Cook ME, DeVoney D, Drake B, Pariza MW, Whigham L, Yang M. Dietary control of immune-induced cachexia: conjugated linoleic acid and immunity. In: Yurawecz MP et al, eds. Advances in conjugated linoleic acid research. Vol 1. Champaign, IL: AOCS Press, 1999:226–37.
45. Hontecillas R, Jobgen SC, Wannemeulher MJ, Bassaganya-Riera J. Differential regulation of colonic PPAR expression by dietary fatty acids. FASEB J2003;17:A1161(abstr).
46. Yang MD, Cook ME. Dietary conjugated linoleic acid decreased cachexia, macrophage tumor necrosis factor-alpha production, and modifies splenocyte cytokines production. Exp Biol Med2003;228:51–8.
47. Ishiguro K, Oku H, Suitani A, Yamamoto Y. Effects of conjugated linoleic acid on anaphylaxis and allergic pruritus. Biol Pharm Bull2002;25;12:1655–7.
48. Whigham LD, Cook EB, Stahl JL, et al. CLA reduces antigen-induced histamine and PGE2 release from sensitized guinea pig tracheae. Am J Physiol Regul Integr Comp Physiol2001;280:R908–12.
49. Whigham LD, Higbee A, Bjorling DE, Park YH, Pariza MW, Cook ME. Decreased antigen-induced eicosanoid release in conjugated linoleic acid-fed guinea pigs. Am J Physiol Regul Integr Comp Physiol2002;282;R1104–12.
50. Chew BP, Wong TS, Shultz TD, Magnuson NS. Effects of conjugated dienoic derivatives of linoleic acid and beta-carotene in modulating lymphocyte and macrophage function. Anticancer Res1997;17:1099–106.
51. Bassaganya-Riera J, Pogranichniy RM, Jobgen SC, et al. Conjugated linoleic acid ameliorates viral infectivity in a pig model of virally induced immunosuppression. J Nutr2003;133:3204–14.
52. Pariza MW, Park Y, Cook ME. Mechanisms of action of conjugated linoleic acid: evidence and speculation. Proc Soc Exp Biol Med2000;223:8–13.
53. Bassaganya-Riera J, Hontecillas R, Beitz D. Colonic anti-inflammatory mechanisms of conjugated linoleic acid. Clin Nutr2002;21:451–9.
54. Banni S, Angioni E, Carta G, et al. Influence of dietary conjugated linoleic acid on lipid metabolism in relation to its anticarcinogenic activity. Champaign, IL: AOCS Press, 1999:307–18.
55. Moya-Camarena SY, Vanden Heuvel JP, Blanchard SG, Leesnitzer LA, Belury MA. Conjugated linoleic acid is a potent naturally occurring ligand and activator of PPARalpha. J Lipid Res1999;40:1426–33.
56. Jiang C, Ting AT, Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature1998;391:82–6.
57. Tontonoz P, Nagy L, Alvarez JGA, Thomazy VA, Evans RM. PPAR gamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell1998;93:241–52.